Alpha-tocopherol beta-oxidation localized to rat liver mitochondria - PubMed (original) (raw)

Alpha-tocopherol beta-oxidation localized to rat liver mitochondria

Debbie J Mustacich et al. Free Radic Biol Med. 2010.

Abstract

Approximately 40% of Americans take dietary supplements, including vitamin E (alpha-tocopherol). Unlike other fat-soluble vitamins, alpha-tocopherol is not accumulated to toxic levels. Rather tissue levels are tightly regulated, in part via increased hepatic metabolism and excretion that could, theoretically, alter metabolism of drugs, environmental toxins, and other nutrients. To date, in vivo subcellular location(s) of alpha-tocopherol metabolism have not been identified. The proposed pathway of alpha-tocopherol metabolism proceeds via omega-hydroxylation to 13'-OH-alpha-tocopherol, followed by successive rounds of beta-oxidation to form alpha-CEHC. To test the hypothesis that alpha-tocopherol omega-hydroxylation occurs in microsomes while beta-oxidation occurs in peroxisomes, rats received daily injections of vehicle, 10 mg alpha-tocopherol, or 10 mg trolox/100 g body wt for 3 days, and then microsomes, mitochondria, and peroxisomes were isolated from liver homogenates. Homogenate alpha-tocopherol levels increased 16-fold in alpha-tocopherol-injected rats, while remaining unchanged in trolox- or vehicle-injected rats. Total alpha-tocopherol recovered in the three subcellular fractions represented 93+/-4% of homogenate alpha-tocopherol levels. In alpha-tocopherol-injected rats, microsome alpha-tocopherol levels increased 28-fold, while mitochondria and peroxisome levels increased 8- and 3-fold, respectively, indicating greater partitioning of alpha-tocopherol to the microsomes with increasing liver alpha-tocopherol. In alpha-tocopherol-injected rats, microsome 13'-OH-alpha-tocopherol levels increased 24-fold compared to controls, and were 7-fold greater than 13'-OH-alpha-tocopherol levels in peroxisome and mitochondrial fractions of alpha-tocopherol-injected rats. An unexpected finding was that alpha-CEHC, the end product of alpha-tocopherol metabolism, was found almost exclusively in mitochondria. These data are the first to indicate a mitochondrial role in alpha-tocopherol metabolism.

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Figures

Fig. 1

Chemical structures of α-tocopherol, α-CEHC and trolox.

Fig. 2

Pristanic acid β-oxidation utilizes both peroxisomes and mitochondria. [15, 17]. *Note the similarities between pristanic acid and the phytyl tail of α-tocopherol.

Fig. 3

Liver and plasma α-tocopherol concentrations in response to subcutaneous (SQ) vehicle, α-tocopherol or trolox injections. α-Tocopherol concentrations in (A) Liver and (B) Plasma. Rats (n = 6/group) received daily SQ injections of vehicle (saline), α-tocopherol or trolox for 3 days. On day 4, following a 12 h fast, rats were killed, blood collected and livers perfused with 0.9% saline (containing 2 U/ml heparin) using a perfusion catheter inserted into the heart. Livers were excised, aliquots frozen in liquid N2 and stored at −80°C. α-Tocopherol concentrations were determined as described in the methods. Liver α-tocopherol was determined per gram of liver and expressed here per total liver in order to facilitate comparison with homogenate and subfraction α-tocopherol levels (Figure 6) and determine recovery of liver α-tocopherol during the homogenation and subfractionation procedure. All values are expressed as mean ± SE, n = 6, with * = p < 0.01 as compared with vehicle-injected rats (see methods).

Fig. 4

Liver and plasma α-CEHC concentrations in response to SQ vehicle, α-tocopherol and trolox injections. α-CEHC concentrations in (A) Liver and (B) Plasma were determined from SQ vehicle-, α-tocopherol and trolox-injected rats, as described in Figure 3 and the methods. Liver α-CEHC was determined per gram of liver and expressed here per total liver in order to facilitate comparison with homogenate and subfraction α-CEHC levels (Figure 7) and determine recovery of liver α-CEHC during the homogenation and subfractionation procedure. All values are expressed as mean ± SE, n = 6, with * = p < 0.01 as compared with vehicle-injected rats (see methods).

Fig. 5

Hepatic homogenate, microsome, mitochondria and peroxisome fraction α-tocopherol concentrations in response to SQ vehicle, α-tocopherol and trolox injections. (A) Total α-tocopherol and (B) Percent homogenate α-tocopherol levels determined for homogenates, microsomes, mitochondria and peroxisomes from SQ vehicle-, α-tocopherol and trolox-injected rats, as described in the methods. Total α-tocopherol is expressed as total nmol per homogenate or subfraction calculated using concentration of α-tocopherol (nmol/g), starting amount of liver (g), total volume of homogenate (ml), volume of homogenate used for fractionation (ml) and final volume used to suspend subfraction pellets. All values are expressed as mean ± SE, n = 6, with * = p < 0.01 as compared with vehicle-injected rats (see methods).

Fig. 6

Hepatic homogenate, microsome, mitochondria and peroxisome fraction 13′-OH-α-Tocopherol levels in response to SQ vehicle, α-tocopherol and trolox injections. (A) Total 13′-OH-α-tocopherol and (B) Percent homogenate 13′-OH-α-tocopherol were determined from SQ vehicle-, α-tocopherol and trolox-injected rats, as described in the methods. Total 13′-OH-α-tocopherol is expressed as total nmol using concentration of 13′-OH-α-tocopherol (nmol/g), starting amount of liver (g), total volume of homogenate (ml), volume of homogenate used for fractionation (ml) and final volume used to suspend subfraction pellets. All values are expressed as mean ± SE, n = 6, with * = p < 0.01 as compared with vehicle-injected rats (see methods)

Fig. 7

Hepatic homogenate, microsome, mitochondria and peroxisome fraction α-CEHC levels in response to SQ vehicle, α-tocopherol and trolox injections. Total α-CEHC is expressed as total nmol calculated using concentration of α-CEHC (nmol/g), starting amount of liver (g), total volume of homogenate (ml), volume of homogenate used for fractionation (ml) and final volume used to suspend subcellular fraction pellets. All values are expressed as mean ± SE, n = 6, with * = p < 0.01 as compared with vehicle-injected rats (see methods).

Fig. 8

Single-quadrupole mass spectral data of α-CEHC in mitochondria and peroxisomes in response to SQ vehicle and α-tocopherol injections. Trolox was added to subcellular fraction samples from vehicle and α-tocopherol injected rats as an internal standard (see methods). α-CEHC and trolox (internal standard) in (A) a representative mitochondrial sample from a vehicle-injected rat, (B) a representative mitochondrial sample from an α-tocopherol-injected rat, (C) a representative peroxisome sample from a vehicle-injected rat and (D) a representative peroxisome sample from an α-tocopherol-injected rat. α-CEHC and trolox were determined by a single-quadrupole LC/MS using an electrospray ionization source, as described in the methods. α-CEHC (mass-to charge ratio (m/z) 277) retention time = 15.42 - 15.46 (solid line). Trolox (m/z 249) retention time = 14.4 - 14.45 (dashed line).

Fig. 9

Proposed pathway of α-tocopherol metabolism in which both peroxisomes and mitochondria play a role

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Alpha-tocopherol beta-oxidation localized to rat liver mitochondria - PubMed (original) (raw)